Evaluation of the pharmacokinetic interaction between ticagrelor and tolbutamide, a cytochrome P450 2C9 substrate, in healthy volunteers.

OBJECTIVES: To assess the effect of ticagrelor on the pharmacokinetics of tolbutamide (a CYP2C9 substrate), and the effect of tolbutamide on ticagrelor pharmacokinetics. METHODS: In this randomized, double-blind, two-period, crossover study, 23 healthy volunteers received either placebo or ticagrelor 180 mg twice daily (b.i.d.) for 9 days, with a single open-label oral dose of tolbutamide 500 mg on Day 5. After washout (14 days), volunteers received the alternate treatment. Plasma concentrations of tolbutamide, 4-hydroxytolbutamide, ticagrelor, and AR-C124910XX were determined for pharmacokinetic analyses. RESULTS: Ticagrelor had no effect on tolbutamide or 4-hydroxytolbutamide pharmacokinetic parameters. The geometric least square mean ratios for maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from Time 0 to infinity (AUC0-∞) were lose to unity, and the 90% confidence intervals (CI) were within the range 0.80 - 1.25 for both tolbutamide and 4-hydroxytolbutamide. The terminal elimination half-life (t1/2), and time to maximal plasma concentrations (tmax) for tolbutamide and its metabolite were unaffected by ticagrelor coadministration. Tolbutamide had no effect on the Cmax, area under the concentration curve over the 2-hour dosing interval (AUC0-τ), t1/2 or tmax of either ticagrelor or AR-C124910XX. Coadministration of ticagrelor and tolbutamide was well tolerated. CONCLUSIONS: These results suggest that ticagrelor does not affect tolbutamide metabolism and is therefore unlikely to affect CYP2C9-mediated metabolism of drugs.

Synthesis and preliminary biological profile of new NO-donor tolbutamide analogues.

We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological characterization of these compounds, including stimulation of insulin release from cultured rat pancreatic ß-cells and in vitro vasodilator and anti-aggregatory activities, is reported.

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats.

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.

In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS.

This study explored the effects of cucurbitacin E (CuE), a bioactive compound from Cucurbitaceae, on the metabolism/pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chromatography-(tandem) mass spectrometry (LC-MS/MS) assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE (1.25-100 µmol L-1) competitively inhibited tolbutamide 4-hydroxylation (CYP2C11) activity only in concentration-dependent manner with a K i value of 55.5 µmol L-1 in vitro. In whole animal studies, no significant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, multiple pretreatments of CuE (200 µg kg-1 d-1, 3 d, i.p.) significantly decreased tolbutamide clearance (CL) by 25% and prolonged plasma half-time (T 1/2) by 37%. Moreover, CuE treatment (50-200 µg kg-1 d-1, i.p.) for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or natural products containing CuE to avoid unnecessary drug-drug interactions.

[The inhibition of CYP2C9 isoenzyme in Cunninghamella blakesleeana AS 3. 910].

AIM: To investigate the variation of CYP2C9 isoenzyme activity in the microbial model in response to inhibitors of CYP2C9. METHODS: Using C. blakesleeana AS 3. 910 as a model strain, the impact of CYP2C9 inhibitors on the metabolites yields of CYP2C9 substrates was determined and the drug-drug interactions among CYP2C9 substrates were evaluated. Liquid chromatography-mass spectrometry was used to analyze biotransformation products. RESULTS: Benzbromarone decreased the yield of 4'-hydroxytolbutamide from 100% to 14.5%; sulfaphenazole decreased the yield of O-demethylindomethacin from 75.2% to 9.9%; valproic acid decreased the yield of 4'-hydroxydiclofenac from 98.6% to 2.7%, separately. Tolbutamide, indomethacin and diclofenac interacted with each other, resulting in the decreased formation of metabolites catalyzed by CYP2C9. CONCLUSION: Three CYP2C9 inhibitors inhibit the activity of CYP2C9 isoenzyme in C. blakesleeana AS 3. 910 differently, and there are drug-drug interactions among CYP2C9 substrates.

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells.

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.

Characterization of protein kinases from bovine parotid glands. The effect of tolbutamide and its derivative on these partially purified enzymes.

1. Four fractions of protein kinase (EC 2.7.1.37) activity (Peak IH, IIH, IIIC and IVC) have been resolved and partially purified from the 100 000 X g supernatant fraction of bovine parotid glands by DEAE-cellulose and phosphocellulose chromatographies. 2. The protein kinases of Peak IH and IIH were adenosine 3',5'-monophosphate (cyclic AMP) -dependent and had similar enzymic properties. The enzyme activities of Peak IIIC and IVC were cyclic-AMP independent, but there were some distinct differences between their properties. The protein kinase in Peak IIIC was activated by 0.2 M NaCl or KCl and phosphorylated casein preferentially as the substrate, utilizing only ATP as a phosphate donor. On the other hand, the protein kinase in Peak IVC was inhibited by univalent salts and preferred phosvitin to casein, utilizing either ATP or GTP as a phosphate donor. 3. Tolbutamide increased the Km value for ATP and the dissociation constant for cyclic AMP, resulting in the inhibition of cyclic-AMP dependent protein kinase activity in the presence of cyclic AMP. Tolbtamide and its carboxy derivative, 1-butyl-3-p-carboxyphenylsulfonylurea, exerted almost no inhibitory effect on either the cyclic-AMP dependent protein kinase activities in the absence of cyclic AMP or on the cyclic-AMP independent protein kinase activities.

Measurement of antidiabetic sulfonylureas in serum by gas chromatography with electron-capture detection.

A method is described for measurement of chlorpropamide, tolbutamide, and the tolbutamide metabolites hydroxymethyltolbutamide and carbotytolbutamide in blood. It consists of formation of the thermally stable methyl-trifluoroacetyl derivatives of chlorpropamide and tolbutamide, and the methyl-heptafluorobutyryl derivatives of hydroxymethyltolbutamide and carboxytolbutamide, which are then analyzed by electron-capture gas chromatography. Measurements of blood levels of the compounds with this method have been verified by quantitation of the same samples using gas chromatography-mass spectrometry in the mass fragmentography mode. Blood level values and beta-phase half-time disappearance rates of chlorpropamide, tolbutamide, hydroxymethyltolbutamide, and carboxytolbutamide were measured in normal volunteers following an oral dose of chlorpropamide or tolbutamide. Blood levels of the four compounds were also determined in a few diabetics receiving continuous daily treatment.

Tolbutamide hydroxylation in humans: lack of bimodality in 106 healthy subjects.

The tolbutamide hydroxylation capacity was studied in 106 healthy unrelated volunteers from the Australian population. Following a 500 mg oral dose of tolbutamide, the ratio of metabolites (hydroxytolbutamide plus carboxytolbutamide) to unchanged tolbutamide excreted in urine from 6 to 12 h post-dose (urinary metabolic ratio, MR) was determined. Metabolic ratio values did not appear bimodally distributed, even following various transformations of the data (i.e. Log10, inverse, Log10 inverse). A poor metabolizer (PM) subject from a previous clinical study, however, could be distinguished (MR value 159) from the above subjects (MR value range 324-3033), particularly from the histogram plot of inverse tolbutamide metabolic ratio. The poor metabolizer's parents had metabolic ratio values (526 and 478) that were at the lower end of the range of metabolic ratios obtained from the population study, and may indicate that they both have a heterozygous genotype and that a recessive form of inheritance is most likely. As the hydroxylations of tolbutamide and phenytoin are closely linked, the incidence of slow tolbutamide metabolizers is likely to be similar to that for phenytoin (about 1:500) and this is consistent with the failure to detect a single poor tolbutamide metabolizer in our random sample of 106 individuals.

Impact of CYP2C9 and CYP2C19 polymorphisms on tolbutamide kinetics and the insulin and glucose response in healthy volunteers.

Tolbutamide is known to be metabolized by cytochrome P450 2C9 (CYP2C9), and the effects of the CYP2C9 amino acid polymorphisms *2 (Arg144Cys) and *3 (Ile359Leu) could be important for drug treatment with tolbutamide and for use of tolbutamide as a CYP2C9 test drug. Tolbutamide pharmacokinetics and plasma insulin and glucose concentrations were studied in 23 healthy volunteers with all six combinations of the CYP2C9 alleles *1, *2 and *3, including two subjects with the combined CYP2C9*1/*1 and CYP2C19*2/*2 genotype. Volunteers received a single oral dose of 500 mg tolbutamide, followed by 75 g oral glucose at 1, 4.5 and 8 h after tolbutamide administration. Pharmacokinetic analysis was performed using a computer program for regression analysis of nonlinear mixed effects models. The mean oral clearances of tolbutamide were 0.97 (95% confidence interval [CI] 0.89-1.05), 0.86 (95% CI 0.79-0.93), 0.75 (95% CI 0.69-0.81), 0.56 (95% CI 0.51-0.61), 0.45 (95% CI 0.41-0.49) and 0.15 (95% CI 0.14-0.16) l/h in carriers of CYP2C9 genotypes 1/*1, *1/*2, *2/*2, *1/*3, *2/*3 and *3/*3, respectively. Tolbutamide pharmacokinetics in carriers of the functionally deficient CYP2C19*2/*2 genotype were not different from those in the CYP2C19 highly active genotype. Elimination in the six CYP2C9 genotype groups could be expressed as the linear combination of three constants (0.05, 0.04, 0.01 h(-1), which were specific to the respective CYP2C9 alleles *1, *2 and *3, thus indicating a co-dominant mode of inheritance. Insulin and glucose concentration-time curves did not change with differing CYP2C9 genotypes. Tolbutamide was confirmed as a substrate of the genetically polymorphic enzyme CYP2C9. The pronounced differences in pharmacokinetics due to the amino acid variants did not significantly affect plasma insulin and glucose concentrations in healthy volunteers.

Effects of CYP2C19 and CYP2C9 genetic polymorphisms on the disposition of and blood glucose lowering response to tolbutamide in humans.

Several recent in-vitro data have revealed that CYP2C19, in addition to CYP2C9, is also involved in the 4-methylhydroxylation of tolbutamide. We evaluated the relative contribution of CYP2C9 and CYP2C19 genetic polymorphisms on the disposition of blood glucose lowering response to tolbutamide in normal healthy Korean subjects in order to reappraise tolbutamide as a selective in-vivo probe substrate of CYP2C9 activity. A single oral dose of tolbutamide (500 mg) or placebo was administered to 18 subjects in a single-blind, randomized, crossover study with a 2-week washout period. Twelve subjects (of whom six were CYP2C19 extensive metabolizer (EM) and six were CYP2C19 poor metabolizer (PM) genotype) were of the homozygous wild-type CYP2C9*1 genotype; the other six subjects were of the CYP2C9*1/*3 and CYP2C19 EM genotype. Pharmacokinetic parameters were estimated from plasma and urine concentrations of tolbutamide and 4-hydroxytolbutamide. Serum glucose concentrations were measured before and after oral intake of 100 g dextrose. In subjects heterozygous for the CYP2C9*3 allele, C(max) and AUC of tolbutamide were significantly greater and the plasma half-life significantly longer than those in homozygous CYP2C9*1 subjects. No pharmacokinetic differences were found between CYP2C19 EM and PM genotype subjects. The estimated AUC of the increase in serum glucose after oral intake of 100 g dextrose was 2.7-fold higher in subjects with the wild-type CYP2C9 genotype than in those with CYP2C9*1/*3, but CYP2C19 genetic polymorphism did not alter the blood glucose lowering effect of tolbutamide. The plasma AUC of 4-hydroxytolbutamide and the ratio of 4-hydroxytolbutamide/tolbutamide did not differ significantly between CYP2C19 PM and EM genotype subjects, while these parameters were about twice as high in subjects with the wild-type CYP2C9 genotype than in heterozygous CYP2C9*3 subjects (P < 0.05). Our results strongly suggest that the disposition and hypoglycemic effect of tolbutamide are affected mainly by CYP2C9 genetic polymorphism, but not by CYP2C19 polymorphism. The in-vivo contribution of CYP2C19 to tolbutamide 4-methylhydroxylation appears to be minor in humans. This suggests that, at least in vivo, tolbutamide remains a selective probe for measuring CYP2C9 activity in humans.

Validation of the tolbutamide metabolic ratio for population screening with use of sulfaphenazole to produce model phenotypic poor metabolizers.

The present study has validated kinetically a convenient method to measure tolbutamide hydroxylation capacity in human beings by use of urinary metabolic ratios. The known in vivo and in vitro inhibitory properties of sulfaphenazole were used to convert control phase subjects to phenotypically "poor" metabolizers of tolbutamide. Six healthy subjects were given a single 500 mg oral dose of tolbutamide with and without sulfaphenazole, 500 mg every 12 hours. Tolbutamide, hydroxytolbutamide, and carboxytolbutamide in urine were determined by newly developed HPLC procedures. Plasma tolbutamide clearance and half-life were measured, as were the metabolic ratio (hydroxytolbutamide + carboxytolbutamide/tolbutamide) in successive 6-hour urine collections. The mean tolbutamide plasma clearance decreased from 0.196 +/- 0.026 ml/min/kg without sulfaphenazole to 0.039 +/- 0.009 ml/min kg with sulfaphenazole, and the mean half-life of tolbutamide increased from 7.28 +/- 0.89 hours to 38.76 +/- 13.30 hours. The metabolic ratio determined in the 6 to 12 hour urine collection period decreased from 794.0 +/- 86.6 to 126.0 +/- 79.3, and this collection period also gave the best separation of subjects between phases. There was a good correlation between tolbutamide plasma clearance and metabolic ratio (rs = 0.853, p less than 0.01, n = 12) and between the percentage decrease in plasma tolbutamide clearance and the percentage decrease in metabolic ratio (r = 0.932, p less than 0.01, n = 6). The tolbutamide urinary metabolic ratio therefore effectively distinguishes tolbutamide hydroxylase activity in "normal" subjects and in those converted to model phenotypically "poor" metabolizers by sulfaphenazole.

Inhibition of tolbutamide metabolism by substituted imidazole drugs in vivo: evidence for a structure-activity relationship.

Tolbutamide has been used as a model drug for an examination of the effects of eleven substituted imidazole compounds on hepatic metabolism in vivo. The 1-substituted compounds 1-methylimidazole, miconazole, clotrimazole and ketoconazole produced marked alterations in tolbutamide kinetics (increased half-life, decreased clearance). However, if there was substitution in the 2- position, irrespective of a substituent on N-1, then the compound did not appear to inhibit metabolism (e.g. 2-methylimidazole, 1,2-dimethylimidazole, methimazole, metronidazole). The 4- substituted compounds, 4-methylimidazole and cimetidine were inhibitors. A structure-activity relationship for the inhibitory actions of the substituted imidazoles is thus evident in vivo.

Tolbutamide as a model drug for the study of enzyme induction and enzyme inhibition in the rat.

The effects of various drugs on the pharmacokinetics of tolbutamide have been examined in the rat. Phenobarbitone pretreatment caused a significant decrease in half life and area under the curve (AUC) and a significant increase in clearance and volume of distribution (Vd). Acute administration of primaquine significantly increased half life and AUC and decreased clearance. In contrast, the related animoquinolone chloroquine, was without effect. Acute administration of cimetidine produced similar changes to primaquine but of lesser magnitude. Formation of the major metabolite hydroxytolbutamide, was markedly enhanced by phenobarbitone and reduced by primaquine and cimetidine. We conclude that due to its single pathway of metabolism, tolbutamide is a good substrate to use when examining pharmacokinetic interactions involving hepatic enzyme induction and inhibition.

Opposite effects of tolbutamide and diazoxide on 86Rb+ fluxes and membrane potential in pancreatic B cells.

The effects of tolbutamide and diazoxide on 86Rb+ fluxes, 45Ca2+ uptake, insulin release and B cell membrane potential have been studied in rat or mouse islets. In the presence of 3 mM glucose, tolbutamide rapidly and reversibly decreased Rb+ efflux from perifused islets and depolarised B cells. The effect on Rb+ efflux was paradoxically more marked with 20 than 100 micrograms/ml tolbutamide, at least in the presence of extracellular calcium. Addition of tolbutamide to a medium containing 6 mM glucose and calcium increased Rb+ efflux transiently with 20 micrograms/ml and permanently with 100 micrograms/ml. The drug also inhibited Rb+ influx in islet cells, but had little effect on Rb+ net uptake. Diazoxide rapidly, steadily and reversibly increased Rb+ efflux in a dose-dependent manner (20-100 micrograms/ml). When 20 micrograms/ml tolbutamide and diazoxide were combined in the presence of 3 mM glucose, only a slight decrease in Rb+ efflux was observed. The depolarisation of B cells normally produced by tolbutamide was markedly reduced and the electrical activity completely suppressed by diazoxide. In the presence of 10mM glucose, diazoxide increased Rb+ efflux from the islets and hyperpolarised B cells. Tolbutamide, tetraethylammonium and quinine reversed the increase in Rb+ efflux the inhibition of Ca2+ uptake and the suppression of insulin release produced by diazoxide. Tolbutamide rapidly reversed the hyperpolarisation and restored electrical activity. It is suggested that the stimulation and inhibition of insulin release by tolbutamide and diazoxide are due to their respective ability to decrease and to increase the K permeability of the B cell membrane. This change in K permeability leads either to depolarisation and stimulation of Ca2+ influx or to hyperpolarisation and inhibition of Ca2+ influx.

Characterization of acetohexamide reductases purified from rabbit liver, kidney, and heart: structural requirements for substrates and inhibitors.

The structural requirements of acetohexamide reductases purified from rabbit liver, kidney, and heart for substrates and inhibitors were examined. Acetohexamide, an oral antidiabetic drug with a ketone group, and analogs of it with various alkyl groups instead of the cyclohexyl group were used as substrates for these three enzymes. The results obtained as to substrate specificity suggested that the nature of the substrate-binding region of the heart enzyme is markedly different from those of the substrate-binding regions of the liver and kidney enzymes. Tolbutamide, which has no ketone group within its chemical structure, strongly inhibited the heart enzyme, whereas it had little ability to inhibit the liver or kidney enzyme. The inhibition of the heart enzyme by tolbutamide was competitive with respect to acetohexamide and uncompetitive with respect to NADPH. Furthermore, tolbutamide analogs with n-pentyl and n-hexyl groups instead of the n-butyl group exhibited very pronounced inhibition of only the heart enzyme. Therefore, it is reasonable to postulate that the heart enzyme, unlike the liver and kidney ones, has a cleft of a strongly hydrophobic nature near its substrate-binding region, and that this hydrophobic cleft plays a critical role in the interaction of the heart enzyme with the cyclohexyl group of acetohexamide.

Inhibition of tolbutamide elimination by cimetidine but not ranitidine.

This study was designed to compare the effects of equivalent therapeutic doses of two H2 antagonists, cimetidine and ranitidine, on tolbutamide pharmacokinetics. Twelve healthy men were given a 1-g oral dose of tolbutamide on three occasions. Subjects were randomly assigned to three treatments in a crossover fashion: cimetidine 1,200 mg/d, ranitidine 300 mg/d, and placebo. Cimetidine significantly increased the tolbutamide area under the plasma concentration-time curve by 20% (range, -5% to 42%), increased the elimination half-life by 17%, and decreased the carboxytolbutamide:tolbutamide plasma ratio from 0.042 to 0.036. Ranitidine did not significantly alter tolbutamide pharmacokinetics.

Determination of tolbutamide hydroxylation in rat liver microsomes by high-performance liquid chromatography: effect of psychoactive drugs on in vitro activity.

A simplified HPLC method for tolbutamide metabolism to hydroxytolbutamide has been used to screen sixty psychoactive drugs for their ability to inhibit rat liver microsomal tolbutamide hydroxylation. One-step extraction with diethyl ether was followed by reconstitution and isocratic HPLC analysis with a binary mobile phase (ammonium phosphate:methanol, 45:55, v/v). Nanogram amounts of hydroxytolbutamide formation were estimated with UV detection at 240 nm. Hydroxytolbutamide formation was linear with incubation times of 40-120 min, but specific activity increased with increases in microsomal protein (0.15-1.10 mg). A differential inhibitory response was demonstrated for tolbutamide and debrisoquine hydroxylation to 5 psychoactive drugs, suggesting that tolbutamide hydroxylation is not dependent on P4502D1. Sixty psychoactive drugs, or drug metabolites, (at 33 microM) were then co-incubated with tolbutamide (at 2.5 and 10.2 microM). Tolbutamide hydroxylation was refractory (< 25% inhibition) to twenty-four of the drugs and only mildly inhibited (25-50% inhibition) by twenty-eight. Two compounds, trans-3-methylfentanyl and flurazepam, produced > 50% inhibition that was independent of tolbutamide concentration. Five of the drugs (methadone, chlorpheniramine, meperidine, 6-monoacetylmorphine and methylphenidate), however, caused greater than 50% inhibition in a competitive manner which suggests these drugs may share an affinity for the substrate binding site for tolbutamide.

High-performance liquid chromatographic assay of tolbutamide and carboxytolbutamide in human plasma.

A high-performance liquid chromatographic method was developed for the simultaneous measurement of tolbutamide and its major metabolite, carboxytolbutamide, in plasma. The assay involves the ether extraction of 1 ml of plasma, using chlorpropamide and an internal standard. The extract is dried, the residue is taken up in acetonitrile, and 5 micro l is injected into a reversed-phase column. The mobile phase consisted of 35% acetonitrile and 65% 0.05 M phosphoric acid buffer (pH 3.9). A fixed-wavelength detector was set at 254 nm. The sensitivity limits for the tolbutamide and carboxytolbutamide assay were 2 and 0.1 microgram/ml, respectively. The ratio of carboxytolbutamide to tolbutamide in plasma obtained from a subject given a 500-mg tolbutamide tablet was 1:20.

Simultaneous determinations of tolbutamide and its hydroxy and carboxy metabolites in serum and urine: application to pharmacokinetic studies of tolbutamide in the rat.

Methods of analysis of tolbutamide (1) and its hydroxylated (2) and carboxylated (3) metabolites in serum and urine based on high-performance liquid chromatography were developed. The separation was performed on a Apex ODS column in the isocratic mode using a mobile phase composed of 22.5% acetonitrile, 77.5% Sorensen phosphate buffer (pH 7.0), and 0.30 mL of tetrabutylammonium phosphate reagent (Pic A). The compounds were detected at 254 mm. The retention times of 3, 2, 1, and the internal standard chlorpropamide were 3.1, 4.1, 14.8, and 10.0 min, respectively. These conditions were suitable for the simultaneous quantitation of 1, 2, and 3 in serum or plasma samples, but not for the determination of metabolites 2 and 3 in urine. For the analysis of 2 and 3 in urine, the mobile phase was modified to 18% acetonitrile, 82% Sorensen phosphate buffer (pH 7.0), and 0.35 mL of Pic A. Under these conditions, the retention times of the carboxy and hydroxylated metabolites and the internal standard salicylic acid were 4.6, 6.7, and 8.1 min, respectively. These methods were applied to study the pharmacokinetics of 1 administered intravenously and intraperitoneally to the rat. Tolbutamide was almost completely recovered as metabolites 2 and 3 in the urine within 24 h.